The central goal of this R01 is to focus on explicitly defining novel antagonist binding pharmacophore on Pregnane X Receptor (PXR). In doing so, additional goals include development of non-toxic azole antagonists that would serve to chemically probe PXR activity and phenotype(s) in different tissues. In silico modeling parameters will continuously be improved as we obtain potent and specific PXR inhibitors. These models could then guide the development of novel small molecule antagonists originating from different chemical entities. The long-term goal is to eventually develop non-toxic antagonists of PXR that can be used as clinical modulators of cancer cell proliferation and drug resistance (e.g., PXR activation induces cancer cell proliferation and drug resistance). It is also hoped that these antagonists will enhance the activity, and minimize the toxicity, of select antineoplastic agents (e.g., tamoxifen, paclitaxel are PXR agonist at concentrations observed at steady-state in humans). Towards this end, we have identified and characterized two novel PXR antagonists, ketoconazole and coumestrol, that specifically disrupt the function of activated (ligand-bound) PXR. In subsequent studies, we have shown that ketoconazole: (i) binds to receptor and disrupts coregulator-receptor interactions in activated PXR;(2) does not displace activating drugs from the ligand-binding pocket of PXR;(iii) retained antagonism of mutant forms of PXR containing ligand-binding pocket filling mutants;and (iv) is unable to antagonize mutant forms of PXR containing alterations in the surface coregulator AF-2 binding site. Thus, we have formulated a model for PXR antagonism in which disruption of function is mediated either by allosteric modification of the receptor or by competition with coregulator binding. We now propose to evaluate this model using structural, molecular, biochemical, and genetic systems to characterize the mechanism by which PXR-directed antagonist ketoconazole and related compounds inhibit receptor activation. PUBLIC HEALTH RELEVANCE: Adopted orphan nuclear receptor, Pregnane X Receptor (PXR), plays a mechanistic role in drug-drug interactions and cancer drug resistance. These functions of the receptor are newly discovered and since this receptor has organ-specific functions, many of its roles in other tissues (e.g., gut) are unknown. PXR is activated by agonists (ligands) and inactivated by antagonists. Our laboratory and co-investigators have taken a lead role in defining novel antagonists of PXR that bind to a region outside the ligand binding pocket. We believe that these antagonists will be necessary to probe PXR function and define all the phenotypes of PXR in mice and humans. In this proposal, we will evaluate our model of antagonism using structural, molecular, biochemical, and genetic systems. These studies will be vital for the generation on non-toxic compounds that antagonize PXR function - a tool necessary to probe PXR function in cells.